Although live attenuated vaccine strains of simian immunodeficiency virus (SIV) have proven highly effective in protecting macaques against challenge with pathogenic SIV strains, little is known about the mechanisms of protective immunity induced by these vaccines. We examined cytotoxic T lymphocyte (CTL) responses against SIV in animals infected with SIVmac239?nef (deficient in nef) or SIVmac239?3 (deficient in nef, vpr and upstream sequences in U3). To enhance detection of SIV-specific CTL activity, we stimulated peripheral blood mononuclear cells with autologous B lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing SIV proteins and subsequently inactivated with psoralen and UV light. Animals chronically infected with SIV239?nef or SIV239?3 mounted vigorous CTL responses against the SIV gag and envelope proteins. This CTL activity was MHC-restricted and mediated by CD8+ T lymphocytes. CTL responses persisted at relatively high levels for more than six years after infection. Limiting dilution precursor frequency assays demonstrated that the frequency of SIV-specific CTL was as high as 234 CTL precursors per 100,000 cells. Animals acutely infected with SIV239?nef developed CTL activity by day 14 after infection, coincident with decreases in viral load. Animals acutely infected with SIV239?3 developed CTL responses within four weeks of infection. Thus, vaccination of juvenile or adult animals with SIV239?nef or SIV239?3 results in the induction of a vigorous CTL response which arises early in the course of infection and persists for years after a single inoculation of virus.